Abstract 1731: Aurora-a mediated phosphorylation of Maf1 is essential for maintaining protein stability and regulating tRNA biosynthesis in HCC
Authors: Ya-Ting Jhan, Yi-Seng Gan, Liang-Yi Hung
Journal: Cancer Research (2023)

Abstract Introduction: Dysregulation of cell cycle plays a crucial role during hepatocarcinogenesis. RNA polymerase III is hyperactive and the products of RNA polymerase III are overexpressed in different types of tumors, including of hepatocellular carcinoma (HCC). Maf1 is an RNA polymerase III repressor that can reduce tRNA biosynthesis via inhibiting the interaction between RNA polymerase and TFIIIB and TFIIIC in nucleus. Maf1 can also inhibit tumor growth and AKT/mTOR signaling pathway via enhancing PTEN expression mTOR can phosphorylate Maf1 at Ser-75, and the phosphorylated Maf1 will be exported into cytosol and then ubiquitinated; therefore, promote protein synthesis and cell cycle progression in HCC. Aurora-A is a serine/threonine kinase and plays a vital role in regulating cell cycle progression. Overexpression of Aurora-A has been observed in HCC. However, the relationship between Maf1 and Aurora-A remains unclear. Materials and Methods: HCC cells were transfected with pGFP-Aurora-A and pHA-Maf1 to investigate their correlation. Alisertib was added to inhibit Aurora-A activity. RT-qPCR was performed to determine the RNA expression of Aurora-A, Maf1, pre-tRNALeu and pre-tRNAiMet. Western blot was performed to analyze expression levels of Maf1 and Aurora-A. Cycloheximide was added to observe the protein stability of Maf1. The protein interaction between Aurora-A and Maf1 was determined by co-immunoprecipitation and proximity ligation assay. The subcellular localization of Maf1 was observed by cell fraction assay and immunofluorescence. The cell proliferation was analyzed by CCK-8. Results: Aurora-A can interact with C domain of Maf1, and promote the cytosolic localization and protein stability of Maf1 in a kinase-dependent manner. Two putative Aurora-A phosphorylation sites of Maf1 were mutated, and we found that one of them can result in a nucleolar localization and a reduced protein stability in Maf1. Surprisingly, we also found that overexpression of the Maf1 mutant (Maf1-MT) cannot suppress tRNA biosynthesis compared to WT Maf1. Maf1-overexpressed HCC cells were more sensitive to Aurora-A inhibitors treatment. Conclusion: Aurora-A can promote the protein stability and cytosolic localization of Maf1 via interacting with its C domain in a kinase-dependent manner in HCC cells. Aurora-A phosphorylation site mutant of Maf1 can promote nucleolar localization but lost the RNA polymerase III suppressor function. In Maf1 overexpressed HCC cells, Aurora-A inhibitor has a higher efficacy in inhibiting cell growth. In HCC patients with high Aurora-A and Maf1 expression, treatment with Aurora-A inhibitors can be a promising strategy in cancer therapy. Citation Format: Ya-Ting Jhan, Yi-Seng Gan, Liang-Yi Hung. Aurora-a mediated phosphorylation of Maf1 is essential for maintaining protein stability and regulating tRNA biosynthesis in HCC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1731.